Expression and function of PTEN in fibroblast-like synoviocytes of rheumatoid arthritis

AIM: investigate the expression of PTEN gene in fibroblast-like synoviocytes (FLS) of rheumatoid arthritis (RA).METHODS: FLS were isolated from synovial tissue obtained from the patients with RA, osteoarthritis (OA) or joint trauma. The mRNA expression of PTEN was detected by RT-qPCR. The protein levels of PTEN, p-Akt (Thr308) and total Akt were determined by Western blotting. The phosphorylation status of Akt was analyzed by the protein ratio of p-Akt (Thr308)/total Akt.RESULTS: The mRNA expression of PTEN was significantly lower in RA-FLS than that in OA-FLS and joint trauma-FLS (P < 0.01), while no statistically significant difference was observed between that in OA-FLS and joint trauma-FLS (P < 0.05). Similarly, the protein expression of PTEN in the RA-FLS was much lower than that in the OA-FLS and joint trauma-FLS (P < 0.05), and there was no difference between the latter 2 groups. Moreover, the phosphorylation level of Akt (Thr308) in the RA-FLS was significantly higher than that in the other 2 control groups (P < 0.01), and that in OA-FLS was much lower than that in the joint trauma-FLS (P < 0.01). Finally, Pearson correlation analysis between the phosphorylation level of Akt (Thr308) and PTEN protein expression in the RA-FLSs showed a significant negative correlation (r=-0.994 5, P < 0.01).CONCLUSION: The mRNA and protein expression of PTEN are both decreased in the RA-FLS, which may contribute to the increased phosphorylation level of Akt (Thr308).     

Effects of fractalkine on nuclear factor-κB activation and endogenous fractalkine mRNA expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis

AIM: To investigate the effects of fractalkine(FKN) on nuclear factor kappa B (NF-κB) activation and endogenous FKN mRNA expression in fibroblast-like synoviocytes (FLS) from the patients with rheumatoid arthritis (RA). METHODS: RA-FLS were gained through tissue culture. Fractalkine at 100 μg/L was used to stimulate RA-FLS for 0 h, 1 h and 2 h. The expression of NF-κB p65 protein in cytoplasm and nucleus was detected by Western blotting, representing the activation of NF-κB in RA-FLS. RA-FLS was stimulated with fractalkine at concentration of 100 μg/L for 0 h, 12 h or 18 h, and the mRNA expression of FKN in RA-FLS was detected by RT-PCR.RESULTS: After stimulated with recombinant human FKN for 1 h, the expression of NF-κB p65 protein in the cytoplasm of RA-FLS was obviously lower than that in RA-FLS without FKN treatment in control group (P<0.05). After stimulated with FKN for 2 h, the expression of NF-κBp65 protein in nucleus was obviously higher than that in RA-FLS of control group (P<0.05). Recombinant human FKN at concentration of 100 μg/L induced endogenous FKN mRNA expression in RA-FLS in a time-dependent manner. The mRNA expression of FKN in RA-FLS obviously increased after stimulated with FKN for 18 h (P<0.05). CONCLUSION: FKN up-regulates the expression of endogenous FKN mRNA, suggesting a positive feedback. FKN can activate the NF-κB and may play an important role in the beginning of joint inflammation, angiogenesis and bone destruction.     

Effects of TREM-2 silencing on migration and invasion abilities of rheumatoid arthritis fibroblast-like synoviocytes

AIM: To investigate the effects of triggering receptor expressed on myeloid cells-2 (TREM-2) silencing on migration and invasion abilities of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). METHODS: Small interference RNA (siRNA) specifically targeting TREM-2 gene was transfected into RA-FLS. The interference efficiency of TREM-2 siRNA on the production of TREM-2 mRNA and protein was determined by RT-PCR and Western blot. The cell activity was assessed by CCK-8 assay. The migration and invasion abilities of RA-FLS were determined by Transwell assay. The releases of MMP-2 and MMP-9 in RA-FLS were analyzed by ELISA. The influence of TREM-2 on PI3K/AKT signal pathway was measured by Western blot. RESULTS: TREM-2 siRNA significantly decreased the mRNA and protein expression of TREM-2. No difference of cell activity between TREM-2 siRNA group and control group was observed. Transwell migration assay showed that RA-FLS through the Transwell membrane in TREM-2 siRNA group were more than the blank control group and the NC-siRNA group. In Transwell invasion assay, RA-FLS through the Transwell membrane in TREM-2 siRNA group were more than the blank control group and the NC-siRNA group. After transfected with TREM-2 siRNA, the MMP-2 secretion and phosphorylation of AKT increased significantly, while the MMP-9 secretion was not changed. CONCLUSION: TREM-2 may play an important role in the migration and invasion of RA-FLS through regulating the activation of PI3K/AKT signal pathway.     

Effect of RICTOR expression on cell viability in rheumatoid arthritis fibroblast-like synoviocytes

AIM：To investigate the effects of RICTOR expression on the viability of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). METHODS：RA-FLS were obtained by tissue culture. Chemically synthesized double-stranded siRNAs targeting RICTOR gene were transfected into RA-FLS by cationic liposome. The nonspecific siRNA was also transfected into the negative control cells. The mRNA expression of RICTOR was detected by RT-qPCR after transfection for 24 h. Western blotting was used to evaluate the inhibitory effects of siRNAs on RICTOR expression after transfection for 48 h and 72 h. The cell viability was examined by MTT assay. RESULTS：Compared with control group, the mRNA expression of RICTOR significantly decreased by 78.36%±3.71% after the transfection of RICTOR siRNA for 24 h. The protein level of RICTOR was also obviously lower in RICTOR siRNA transfection group than that in control group after transfection for 48 h (decreased by 92.48%±6.14%) and 72 h (decreased by 98.57%±1.40%). Knock-down of RICTOR in RA-FLS for 72 h markedly decreased the cell viability. CONCLUSION：Transfection of RICTOR siRNA reduces the viability of RA-FLS, indicating that mTORC2 may be required for the survival of RA-FLS.     

Sinomenine induces apoptosis of human rheumatoid arthritis fibroblast-like synoviocytes via miR-23b-3p/FGF9 signaling pathway

AIM To investigate the effect of sinomenine （SIN） on the apoptosis of human rheumatoid arthritis （RA） fibroblast-like synoviocytes （FLS） and its molecular mechanism. METHODS Human RA FLS were isolated and cultured. The cells treated with lipopolysaccharide （LPS） at 100 mg/L was recorded as LPS group. The cells treated with SIN at 3.2 mmol/L and LPS at 100 mg/L were recorded as LPS+SIN group. The cells without any treatment served as blank group. The cells transfected with miR-con, miR-23b-3p, si-con and si-fibroblast growth factor 9 （FGF9） and treated with 100 mg/L LPS were recorded as LPS+miR-con group, LPS+miR-23b-3p group, LPS+si-con group and LPS+si-FGF9 group, respectively. The anti-miR-con, anti-miR-23b-3p, pcDNA and pcDNA-FGF9 were also transfected into RA FLS, and then the cells were treated with SIN at 3.2 mmol/L and LPS at 100 μg/mL. These cells were recorded as LPS+SIN+anti-miR-con group, LPS+SIN+anti-miR-23b-3p group, LPS+SIN+pcDNA group, LPS+SIN+pcDNA-FGF9 group, respectively. The cell viability was measured by CCK-8 assay. The number of colonies was accessed by colony formation experiment. The protein levels of FGF9, cleaved caspase-3, Bax and Bcl-2 were determined by Western blot. The apoptosis was analyzed by flow cytometry. The expression of miR-23b-3p and FGF9 mRNA was detected by RT-qPCR. Dual-luciferase reporter assay was used to verify the targeting relationship between miR-23b-3p and FGF9. RESULTS Treatment with SIN promoted LPS-induced apoptosis of RA FLS, inhibited cell proliferation, up-regulated miR-23b-3p expression, and down-regulated FGF9 expression. miR-23b-3p targeted FGF9. Over-expression of miR-23b-3p or silencing of FGF9 inhibited LPS-induced proliferation and enhanced apoptosis of the RA FLS. Interfering with miR-23b-3p or over-expression of FGF9 reversed the effects of SIN on the proliferation and apoptosis of LPS-induced RA FLS. CONCLUSION Sinomenine induces RA FLS apoptosis and inhibits cell proliferation through miR-23b-3p/FGF9 signaling.     

Relationship between anti-CCP antibody and migration or invasion ability of fibroblast-like synoviocytes from rheumatoid arthritis patients

AIM:To investigate the relationship between anti-cyclic citrullinated peptide (anti-CCP) antibody and migration or invasion ability of fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. METHODS:FLS was isolated from 22 patients with active RA, 12 patients with osteoarthritis (OA) and 6 patients with joint injuries without arthritis history. The migration and invasion abilities were assessed by Transwell assay. RESULTS:Among the patients of RA, OA and normal controls, the numbers of migratory cells were (29.33±10.93), (9.28±7.87) and (7.00±4.07)/field, respectively, and the numbers of invasive cells were (14.35±7.67), (3.96±4.37) and (3.08±1.03)/field, respectively. The numbers of migratory and invasive cells were higher in RA than those in the other groups. The migration and invasion abilities of RA FLS from anti-CCP antibody-positive RA patients were significantly increased as compared with anti-CCP antibody-negative RA patients. Correlation analysis revealed that the migration and invasion abilities of RA FLS were correlated with the positive and high titer of anti-CCP antibody.CONCLUSION: The migration and invasion abilities of FLS from anti-CCP antibody-positive RA patients are increased. Anti-CCP antibody is possibly correlated with the increased migration and invasion abilities of RA FLS.     

Rosiglitazone inhibits osteoclastogenesis in rheumatoid arthritis by down-regulating RANKL expression and suppressing ERK phosphorylation

AIM: To investigate the effects of rosiglitazone on fibroblast-like synoviocyte (FLS)-induced osteoclastogenesis in rheumatoid arthritis (RA) and the related mechanism. METHODS: RA-FLS were cocultured with peripheral blood monocytes from healthy volunteers in the presence of macrophage colony-stimulating factor (M-CSF) and rosiglitazone. Osteoclasts were assayed by tartrate-resistant acid phosphatase (TRAP) staining. Resorption lacunae area was identified by toluidine blue staining and quantified by image analysis software. The mRNA expression of RANKL and OPG was evaluated by real-time PCR, and the protein levels of RANKL, OPG, p-ERK, p-p38 and p-JNK were measured by Western blot. RESULTS: Compared with control group (without rosiglitazone treatment), rosiglitazone at concentration of 15 μmol/L significantly decreased the number of osteoclasts (P<0.01) and resorption lacunae area (P<0.05). The expression of RANKL at mRNA and protein levels was significantly down-regulated by rosiglitazone at concentration of 15 μmol/L, while the mRNA and protein expression of OPG was up-regulated (P<0.01). Rosiglitazone (15 μmol/L) significantly decreased the protein level of p-ERK (P<0.05), but not the protein level of p-p38 or p-JNK (P>0.05). CONCLUSION: Rosiglitazone inhibits RA-FLS-induced osteoclast formation and its resorption activity by down-regulating RANKL expression and ERK phosphorylation, suggesting that rosiglitazone may inhibit RA osteoclastogenesis and bone resorption.     

Prolactin induces interleukin-6 expression in PBMCs of rheumatoid arthritis patients

ATM: To investigate the correlation between serum prolactin (PRL) levels and disease activity in rheumatoid arthritis (RA) patients, and the regulatory role of PRL in interleukin-6 (IL-6) release from peripheral blood mononuclear cells (PBMCs), and to explore the MAPK-related mechanism of IL-6 release in PBMCs. METHODS: The clinicopathologic and hematologic parameters of 40 new-onset RA patients in the Department of Rheumatology of our hospital between March and September 2015 were collected. Chemilumineseent immunoassay (CLIA) was used to detect the serum PRL levels in the 40 RA patients and 20 healthy controls. The levels of IL-6 secretion by the PBMCs were evaluated using ELISA. Quantitative real-time PCR was employed to examine the mRNA expression of prolactin receptor (PRLR). MAPK pathway protein p-p38 levels were evaluated by Western blot. RESULTS: Serum PRL level in the RA patients was significantly higher than that in the healthy controls (P<0.01). Serum PRL level in active RA patients was significantly higher than that in inactive RA patients (P<0.01). Serum PRL level was positively correlated with DAS28, ESR and CRP (P<0.01). The expression of PRLR in the PBMCs was markedly increased in the RA patients than that in the healthy samples (P<0.01). Exposure of the PBMCs to PRL in the culture increased the release of IL-6, which was abolished by PRLR gene silencing or blocking the MAPK pathway.CONCLUSION: Serum PRL level is related to DAS28, ESR and CRP of RA patients and could be used as a predictor of disease activity. PRL/PRLR-p38 MAPK-IL-6 pathway may play a central role in the pathogenesis of RA.     

Effect of protease inhibitor bortezomib on treatment of rheumatoid arthritis by affecting IL-33/ST2 signaling pathway

AIM To investigate the effect of bortezomib, a protease inhibitor, on the treatment of rheumatoid arthritis （RA） and it mechanism, based on interleukin-33 （IL-33）/suppression of tumorigenicity 2 （ST2） signaling pathway. METHODS A total of 40 Wistar rats were randomly divided into 4 groups： control group, model group, and low- and high-dose bortezomib groups, with 10 rats in each group. In addition to control group, the rats in other groups were used to construct RA model. Bortezomib was given intraperitoneally at 0.2 mg/kg and 0.5 mg/kg in low- and high-dose bortezomib groups, respectively, while the rats in control group and model group were injected with the same amount of saline, once a day for 21 d. The general situation of the rats in each group was observed, the swelling degree of the foot was calculated, and the inflammation score was evaluated. HE staining was used to observe the pathological changes of ankle joint. The automatic biochemical analyzer was used to detect blood hemoglobin content, the total number of platelets （PLT）, serum creatinine （SCr） level and blood urea nitrogen （BUN） level. The serum levels of IL-6, tumor necrosis factor-α （TNF-α）, IL-33 and ST2 were measured by ELISA. The protein expression of IL-33 and ST2 in ankle tissues of each group was determined by Western blot. RESULTS On the 7th, 14th and 21th days after modeling, compared with control group, the degree of paw swelling in model group was significantly increased （P<0.05）. Compared with model group, the swelling degree of paw in low- and high-dose groups was decreased （P<0.05）. At the end of administration, compared with control group, the synovial cells in model group were increased and in disorder, with a lot of inflammatory exudates in the articular cavity, and the inflammatory score, the levels of PLT, SCr and BUN, the serum levels of IL-6, TNF-α, IL-33 and ST2, and the protein expression of IL-33 and ST2 in ankle tissues were significantly increased （P<0.05）. Compared with model group, the inflammatory exudates in the articular cavity of the rats in low- and high-dose bortezomib groups were decreased, and the inflammatory score, the levels of PLT, SCr and BUN, the serum levels of IL-6, TNF-α, IL-33 and ST2, and the protein expression of IL-33 and ST2 in ankle tissues were decreased （P<0.05）. CONCLUSION Bortezomib may reduce the inflammation and swelling of the joints in RA rats by regulating the IL-33/ST2 signaling pathway.     

Effects of microRNA-16 on proliferation,invasion and cytokine secretion of synovial fibroblasts from rheumatoid arthritis patients

AIM:To investigate the effect of microRNA-16 (miR-16) on the proliferation, invasion and cytokine secretion of rheumatoid arthritis (RA) synovial fibroblasts (RASFs) from the RA patients. METHODS:miR-16 mimic and miR-16 inhibitor were synthesized, and then Transfected into RASFs isolated from RA patients with lipofectamine. MTT assay, Transwell chamber and flow cytometry were used to determine the effect of miR-16 on proliferation, invasion and apoptosis of RASFs. The expression of matrix metalloproteinase 3/13 (MMP3/13) and interleukin 1β (IL-1β) was measured by RT-PCR and Western blotting. RESULTS:The proliferation and invasion of RASFs were significantly inhibited by miR-16 mimic. The result of flow cytometry demonstrated that miR-16 had no effect on apoptosis of RASFs. Furthermore, miR-16 down-regulated the expression of MMP3/13 and IL-1β. CONCLUSION: miR-16 plays an important role in the development of RA and may inhibit the proliferation and invasion of RASFs through down-regulating the expression of MMP3/13 and IL-1β.     

Effect of Tripterygium hypoglaucum Hutch on MMP-13 protein expression and IL-12, IL-23 and IL-37 levels in serum and foot paws of rats with collagen-induced arthritis

AIM: To investigate the effect of tripterygium hypoglaucum Hutch (THH) on collagen-induced arthritis (CIA) in rats and its possible mechanism. METHODS: SD rats were randomly divided into normal group and CIA group. The rat model of type Ⅱ CIA was successfully established and the model rats were randomly divided into 4 different groups: model group, dexamethasone group, THH (200 mg/kg) group, and THH (400 mg/kg) group. The contents of IL-12, IL-23 and IL-37 in the serum and foot paws of the CIA rats were detected by ELISA. The histopathological changes of the skin of the food paws were observed by HE staining. The protein expression of MMP-13 was determined by Western blot. The MMP-13 activity in the foot paws was detected by fluorescence labeling method. RESULTS: Compared with CIA group, THH at dose of 400 mg/kg significantly reduced the weight loss in type Ⅱ CIA rats (P<0.01). THH at dose of 400 mg/kg obviously decreased the contents of IL-12 by 28.31%, IL-23 by 41.57% in the serum and IL-12 by 30.78%, IL-23 by 39.46% in the foot paws, while IL-37 was significantly increased by 79.43% in the serum and 75.78% in the foot paws (P<0.01). The pathological changes of the subcutaneous tissues were improved by treating with THH (400 mg/kg). The protein expression of MMP-13 was significantly decreased by 31.82% (P<0.01), and the MMP-13 activity was also reduced. THH at dose of 200 mg/kg had no obvious improvement on the above indexes. CONCLUSION: THH has significant inhibitory effect on rat CIA by reducing the content of proinflammatory cytokines IL-12 and IL-23, increasing the content of anti-inflammatory factor IL-37, inhibiting inflammatory cell infiltration and vascular proliferation, and attenuating the protein expression of MMP-13 and MMP-13 activity in rats.     

Relationship between IL-1 gene polymorphism and severity of rheumatoid arthritis and secretion of IL-1

AIM: To determine the influence of interleukin-1α(IL-1α) and β(IL-1β) gene polymorphisms on rheumatoid arthritis(RA) disease severity and secretion of IL-1β. METHODS: The study included 136 RA patients and 102 healthy controls. PCR-RFLP was used to detect site mutation at IL-1 gene. Meanwhile the IL-1β was also measured in the supernatant of the cultured and stimulated peripheral blood mononuclear cells(PBMC). RESULTS: No difference in the allele frequencies or genotypes of the IL-1α gene polymorphisms was found between the controls and RA patients.IL-1β allele 2 was overrepresented in patients with erosive RA but not in nonerosive patients. The patients with IL-1β allele 2 had a higher swollen joint index, higher tender joint index and erythrocyte sedimentation rate than those without IL-1β allele 2.The IL-1β in supernatant of stimulated PBMC from patients with IL-1β allele 2 had a higher level than that from those without allele 2. CONCLUSION: IL-1 gene polymorphisms may influence the occurrence of RA. Detection of IL-1β allele 2 have a potential prognostic value in RA.     

Effect of miR-23b-3p on viability and apoptosis of rheumatoid arthritis synovial fibroblasts by targeting XIAP

AIM:To investigate the effect of microRNA-23b-3p (miR-23b-3p) on the viability and apoptosis of rheumatoid arthritis synovial fibroblasts by targeting X-linked inhibitor of apoptosis protein (XIAP). METHODS:The expression of miR-23b-3p and XIAP was detected by RT-qPCR. The TargetScan was used to predict the targeting regulatory relation between miR-23b-3p and XIAP, and then the regulatory relation was confirmed by dual-luciferase reporter assay. After the miR-23b-3p mimic and inhibitor were transfected into the cells, the expression of miR-23b-3p and XIAP was detect by RT-qPCR. The effect of miR-23b-3p on the viability and apoptosis was measured by CCK-8 assay and flow cytometry. The protein expression levels of Ki67 and Bcl-2 were determined by Western blot. RESULTS:The expression level of miR-23b-3p was down-regulated significantly (P<0.05), and XIAP was up-regulated significantly in rheumatoid arthritis synovial fibroblasts (P<0.05). The miR-23b-3p mimic significantly inhibited XIAP expression and the cell viability, promoted the apoptosis, and down-regulated the expression of Ki67 and Bcl-2 (P<0.05). The effects of miR-23b-3p inhibitor were the opposite. CONCLUSION:miR-23b-3p inhibits the viability and promotes apoptosis of rheumatoid arthritis synovial fibroblasts by targeting XIAP.     

IL-1β promotes differentiation of naïve T cells into Th22 cells and its expression in non-small cell lung cancer

AIM:To investigate the mechanism of interleukin-1β (IL-1β) promoting the transformation of naïve T cells into Th22 cells and the correlation of its peripheral blood expression in non-small cell lung cancer patients. METHODS:CD4⁺ naïve T cell magnetic bead sorting kit was used to isolate the peripheral blood mononuclear T cells from healthy people. Transforming growth factor-β (TGF-β) and IL-2 were added to promote differentiation and proliferation. IL-1β was used to induce differentiation into Th22 cells. The proportion of CD4⁺ IL-22⁺ T cells was analyzed by flow cytometry, and the expression of IL-22 was detected by ELISA. We selected 60 cases of non-small cell lung cancer patients in our hospital, including 18 in I phase, 20 in Ⅱ phase, 13 in Ⅲ phase and 9 in IV phase, as well as 25 healthy persons. The proportion of Th22 (CD4⁺ IL-22⁺) cells in peripheral blood was detected by flow cytometry, and the serum levels of IL-1β and IL-22 were measured by ELISA. RESULTS:IL-1β induced the transformation of naïve T cells into Th22 cells and promoted the secretion of IL-22 (P<0.05). The proportion of Th22 cells and the IL-22 and IL-1β levels in peripheral blood of the patients with non-small cell lung cancer were higher than those in healthy subjects, and correlated with the clinical stage. CONCLUSION:IL-1β induces the differentiation of Th22 cells and the expression of IL-22. The levels of IL-1β and IL-22 are related to the progression of non-small cell lung cancer, which may be involved in immunosuppression and promote the occurrence of non-small cell lung cancer.     

Effects of lipoxin A4 on proliferation and IL-2/IL-2 receptor expression in Jurkat T cells

AIM: To investigate the effect of lipoxin A4 on the proliferation and IL-2 production in Jurkat T cells. METHODS: Jurkat T cells were activated in vitro with anti-CD3 (2 mg/L) and anti-CD28 (2 mg/L) antibodies in the absence or presence of lipoxin A4 (0.1 nmol/L-100 nmol/L) for 24 h, then ［3H］-TdR was added into the medium and radioactivities were measured by scintillation counting. The concentrations of IL-2 in the supernants were determined by ELISA. Cells were harvested and the expression of CD25 was assessed by FCM. For analysing the cell cycle, the cells were stained with PI and DNA contents were detected by FCM. RESULTS: Lipoxin A4 suppressed the proliferation of anti-CD3 and anti-CD28 antibodies activated Jurkat cells in a dose-dependent manner, which was associated with reduced proportion of S phase cells. Furthemore, lipoxin A4 significantly inhibited the production of IL-2 but had no obvious effect on CD25 expression. CONCLUSION: Lipoxin A4 can suppress proliferation of activate Jurkat cells and IL-2 production, through which lipoxin A4 might negatively regulate immune response.     

Effect of tetramethylpyrazine on paw MMP-13 protein expression and serum VEGF,IL-17 and IL-23 levels in rats with collagen-induced arthritis

AIM：To investigate the mechanism of tetramethylpyrazine (TMP) in treating collagen-induced arthritis (CIA) in rats. METHODS：The rat model of type II collagen-induced arthritis was established and the rats were randomly divided into normal control group, CIA group and TMP treatment group. The protein expression of matrix metalloproteinase 13 (MMP-13) in paw tissues was detected by immunohistochemical staining and Western blotting. The histopathological changes of the skin in the rat paws were observed with HE staining. The levels of vascular endothelial growth factor (VEGF), interleukin 17 (IL-17) and interleukin 23 (IL-23) in the serum of CIA rats were detected by ELISA. RESULTS：Compared with CIA group, TMP at a dose of 100 mg/kg significantly reduced the weight loss in CIA rats and decreased the protein expression of MMP-13 by 31.82%. TMP also attenuated the pathological changes of the paw subcutaneous tissues. TMP obviously decreased the levels of VEGF by 33.33%, IL-17 by 27.40% and IL-23 by 33.33% in the serum. CONCLUSION：TMP significantly inhibits the development of CIA by decreasing the protein expression of MMP-13, inhibiting the inflammatory cell infiltration and vascular proliferation, and reducing the production of VEGF, IL-17 and IL-23 in CIA rats.     

Effects of IL-6 and AG490 on growth of diffuse large B-cell lymphoma and Burkitt lymphoma transplanted into nude mice

AIM: To observe the effects of interleukin-6 (IL-6) and AG490 on diffuse large B-cell lymphoma (DLBCL) and Burkitt lymphoma (BL) transplanted into nude mice, and to explore the effects of STAT3 activation on growth of these kinds of lymphoma in nude mice and its related mechanisms. METHODS: The nude mouse models with DLBCL and BL were established by transplantation with OCI-LY8 cells and Raji cells, respectively, and were divided into 3 groups:control group, IL-6 group and AG490 group. The body weight of mice and tumor size were measured. Western blot and immunohistochemical staining were used to detect the protein levels of p-STAT3, survivin and vascular endothelial growth factor (VEGF), and real-time PCR was used to detect the mRNA expression of survivin and VEGF. RESULTS: The tumorigenic rate of 2 kinds of tumor cell lines in nude mice was 83.3% (25/30) totally. The tumorigenicity of OCI-LY8 cells (66.7%, 10/15) was significantly lower than that of Raji cells (100%, 15/15) (P<0.05). The tumor size and body weight on days 9 and 10 in IL-6 group increased as compared with the control group, and the total difference value of tumor size between day 1 and day 10 in IL-6 group was obviously larger than that in control group (P<0.05). The positive protein of p-STAT3 was found in the nucleus, while the positive expression of survivin and VEGF was found in the cytoplasm. As compared with control group, the expression of survivin and VEGF was significantly increased (P<0.05), while the protein level of p-STAT3 was not significantly increased in IL-6 group of DLBCL. The protein levels of p-STAT3 and VEGF were significantly decreased (P<0.05), while the expression of survivin did not significantly decreased in AG490 group of DLBCL. The p-STAT3 and VEGF levels significantly increased (P<0.05) in IL-6 group of BL, while the levels of 3 kinds of proteins significantly deceased (P<0.05) in AG490 group of BL, as compared with control group. No statistical difference of mRNA expression of survivin and VEGF among IL-6, AG490 and control groups was observed. CONCLUSION: IL-6 and AG490 affect the growth of DLBCL and BL through activation of STAT3 pathway. The activated STAT3 participates in pathogenesis and progress of DLBCL and BL by up-regulating the expression of survivin and VEGF.     

Inhibitory effects of paeonol on keratinocyte viability,cytokines secretion stimulated by IL-17A via STAT3 signaling pathway

AIM To observe the effect of paeonol on interleukin-17A （IL-17A）-induced human keratinocyte viability, cytokine secretion, and related signal transduction pathways. METHODS In vitro HaCaT cells stimulated by IL-17A （200 μg/L） were co-cultured with paeonol （200 mg/L and 100 mg/L） for 24 h. The cell viability was measured by CCK-8 assay. The Th1/Th2/Th17 cytokine （including IL-6, etc.） levels were measured by cytometric bead array assay. The IL-23 level was measured by ELISA. The mRNA expression of IL-23, IL-6, CXCL2, CXCL8, CCL20 and STAT3 was detected by real-time PCR, and Western blot was used to determine the protein levels of STAT3 and ERK1/2. RESULTS Paeonol significantly inhibited IL-17A-induced HaCaT cell viability （P<0.05）, as well as reduced IL-6 level. Meanwhile, paeonol decreased mRNA levels of IL-23, CXCL2, CXCL8, and CCL20. Paeonol also inhibited the expression of STAT3 at mRNA and protein levels. However, no significant effect of paeonol on ERK1/2 protein expression was observed. CONCLUSION Paeonol inhibits HaCaT cell viability and cytokine secretion induced by IL-17A, and its mechanism might be related to STAT3 singaling pathway.     

Effects of sodium valproate on RPMI8226 and U266 cell proliferation and IL-6/JAK/STAT signaling pathway

AIM: To investigate the effects of sodium valproate (VPA) on the proliferation of multiple myeloma cell lines RPMI8226 and U266 and the regulation of IL-6/JAK/STAT signaling pathway. METHODS: The cells were treated with different concentrations of VPA for 12 h and 24 h. The growth of RPMI8226 cells and U266 cells was detected by MTT assay. Apoptotic rates and cell cycle were analyzed by flow cytometry. The mRNA expression of STAT3, STAT5 and STAT target genes Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF was measured by RT-PCR. Western blotting analysis was used to determine the total proteins and protein phosphorylation levels of JAK2 and STAT5. RESULTS: VPA inhibited the growth and induced the apoptosis of RPMI8226 cells and U266 cells in a concentration- and time-dependent manner. The levels of IL-6 in the culture supernatants of RPMI8226 cells and U266 cells treated with VPA were significantly higher than that in negative control group. VPA down-regulated the mRNA expression of STAT3, STAT5, Bcl-xL, Mcl-1, c-Myc, CCND1 and VEGF. After treated with VPA, the protein levels of p-JAK2, JAK2, p-STAT5 and STAT5 in RPMI8226 cells and U266 cells were significantly lower than those in control group. CONCLUSION: VPA inhibits the proliferation of PRMI8226 cells and U266 cells in vitro. The modulation of IL-6/JAK/STAT signaling pathway may be involved in its potential mechanisms.